Research in the past few decades has revealed many key factors in erythropoietin (Epo) and Rac GTPase signaling pathways that are important for the development of various red blood cell related diseases. However, the pathogenesis of many other erythroid disorders, such as congenital dyserythropoietic anemias, Fanconi anemia, sideroblastic anemia, unexplained anemia of the elderly, ineffective erythropoiesis in beta-thalassemia, and myelodysplastic syndromes, remain unclear. These diseases often show unexplained defects in cell differentiation, survival, and actin dynamics that mimic disruptions of Epo and/or Rac GTPase pathways. However, no direct defects in Epo or Rac GTPase pathways have been reported to date in these diseases. An open question in the field is whether there are any proteins or pathways that link Epo and Rac GTPase pathways to regulate terminal erythropoiesis, which could be involved in the pathogenesis of aforementioned diseases. In this effort, we discovered that pleckstrin-2 (plek2) plays a critical role in different stages of terminl erythropoiesis through the interaction with cofilin. Our preliminary data and reports from other groups indicated that plek2 is closely interrelated with Epo and Rac GTPase pathways to regulate erythroid cell differentiation, survival and actin dynamics to form a global regulatory network in erythropoiesis. In this project, we will determine the roles of plek2 as a functional node in erythropoiesis in vitro and in vivo using our well-established mouse fetal erythroblast culture system and various mouse models. In Aim 1, we will use plek2 knockout mouse model, which showed macrocytic anemia and fetal anemia, to determine the functions of plek2 in erythropoiesis in vivo under steady state and stress conditions. The role of plek2 in the pathogenesis of beta-thalassemia will also be tested using Hbbth1/th1 mice as a direct red cell disease model for plek2. In Aim 2, we will determine the mechanisms by which plek2 is regulated by Epo signaling pathways transcriptionally or through post-translational modifications. In Aim 3, we will determine the extent to which plek2-cofilin and Rac GTPase pathways are reciprocally required in different stages of terminal erythropoiesis. Successful accomplishment of these independent but interconnected aims will reveal a central role of plek2 in regulating cell differentiation, survival and actin dynamics in erythropoiesis, which could provide novel insights into the pathogenesis and therapeutic management of red cell related diseases with unclear etiology.